Fabrication of multifunctional ZnO@tannic acid nanoparticles embedded in chitosan and polyvinyl alcohol blend packaging film

The current study explores biodegradable packaging materials that have high food quality assurance, as food deterioration is mostly caused by UV degradation and oxidation, which can result in bad flavor and nutrition shortages. Thus, new multifunctional zinc oxide nanoparticles/tannic acid (ZnO@TA) with antioxidant and antibacterial activities were incorporated into polyvinyl alcohol/chitosan (PVA/CH) composite films with different ratios (1%, 3%, and 5% based on the total dry weight of the film) via a solution blending method in a neutral aqueous solution. Additionally, ZnO nanoparticles have unique antibacterial mechanisms through the generation of excessive reactive oxygen species (ROS) that may lead to intensify pathogen resistance to conventional antibacterial agents. Thus, minimizing the negative effects caused by excessive levels of ROS may be possible by developing unique, multifunctional ZnO nanoparticles with antioxidant potential via coordination bond between tannic acid and ZnO nanoparticles (ZnO@TA). ZnO@TA nanoparticles were examined using Fourier-transform infrared (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The effect of the incorporation of ZnO@TA nanoparticles on the barrier, mechanical, thermal, antioxidant, antimicrobial, and UV blocking characteristics of chitosan/polyvinyl alcohol (ZnO@TA@CH/PVA) films was investigated. The lowest water vapor and oxygen permeability and the maximum antioxidant capacity% are 31.98 ± 1.68 g mm/m2 kPa day, 0.144 ± 5.03 × 10–2 c.c/m2.day, and 69.35 ± 1.6%, respectively, which are related to ZnO@TA(50)@CH/PVA. Furthermore, ZnO@TA(50)@CH/PVA film exhibits the maximum UV shielding capacity of UVB (99.994). ZnO@TA(50) @PVA/CH films displayed better tensile strength and Young`s modulus of 48.72 ± 0.23 MPa and 2163.46 ± 61.4 MPa, respectively, than the other film formulations. However, elongation % at break exhibited the most reduced value of 19.62 ± 2.3%. ZnO@TA@CH/PVA film exhibits the largest inhibition zones of 11 ± 1.0, 12.3 ± 0.57, and 13.6 ± 0.57 mm against Staphylococcus aureus, Aspergillus flavus, and Candida albicans, respectively. In accordance with these results, ZnO@TA@CH/PVA films could be utilized for food preservation for the long-term.


Materials and methods
Zinc acetate dihydrate 98% was purchased from Merck.Chitosan medium molecular weight with deacetylation percentage of 75-85% deacetylated, poly (vinyl alcohol) (PVA) molecular weight 89.000-98.000(+ 99%-degree hydrolysis) were acquired from Merck.Tannic acid (Mw 1701.2 g/mol) was purchased from Sigma Aldrich.The solvent of chitosan was 98% acetic acid which purchased from a local company of El Naser (Egypt).

Preparation of ZnO nanoparticles
ZnO nanoparticles were prepared as described.A stock solution of (0.1 M) zinc acetate dihydrate was vigorously stirred into methanol (50 mL).The pH of the mixture was then maintained at 8 by adding (0.2 M) NaOH in methanol (25 mL).The mixture was sealed in a Teflon-lined stainless-steel autoclave at 80 °C for 10 h.The white precipitate was washed with methanol, filtered, and then dried in a vacuum oven at 60 •C for 6 h. 16.
Preparation of ZnO@tannic acid nanoparticles A wet chemical process with tannic acid was used to provide ZnO nanoparticles with antioxidant functionality, as follows.Subsequently, 20 mg of ZnO nanoparticles were dispersed in 1 mL of EtOH.Then the above solution was poured to a solution of tannic acid/EtOH (2.08 × 10 −5 M), vigorously stirred for 24 h.The resulted pale brown precipitate was washed several times in EtOH and dried at 60 •C 17 .
The average particle size and Zeta-potential of ZnO@TA The average particle size and Zeta-potential of ZnO@TA was determined with the aid of a dynamic light scattering (DLS) PS analyzer (ZetaSizer Nano ZS ZEN3600 from Malvern Instrument; Particle Sizing Systems, Inc.Santa Barbara, CA), every measurement was done at 25°C.The samples were serially diluted 100 times using deionized water.
Fabrication of ZnO/TA@ CH/ PVA films CH/PVA film was used as a control, and (0.5 g) of PVA was dissolved in 50 mL of hot, deionized water while being stirred.Chitosan solution (0.5 g/50 mL of 2% acetic acid) was added to PVA solution under stirring with addition of 0.1 g glycerol.After ultrasonic treatment, the mixed solution was then poured into Teflon dishes and let to air dry.ZnO/TA were added with different loadings (1%, 3%, and 5%) based on CH/PVA (1 g) which are equivalent to 10 mg, 30 mg, and 50 mg ZnO/TA, and solid content of all films was kept constant to get a constant thickness (0.166 ± 5.77 × 10 −3 mm) as illustrated in Table 1.The prepared films are referred to CH/PVA, ZnO@ TA(10)@CH/PVA, ZnO@TA(30)@CH/PVA, and ZnO@TA(50)@CH/PVA.

Fourier-transform infrared (FTIR)
The chemical structures of ZnO NPs, TA, ZnO@TA, CH/PVA, and ZnO@TA@CH/PVA films were determined by Fourier Transformation Spectroscopy (FTIR) spectra at room temperature using the Attenuated Total Reflection (ATR) unit attached with FTIR-Vertex 70 Bruker, Germany, in the range of 4000-400 cm -1 .
Using the Scherer equation (Eq. 1) and the diffraction intensity of the (101) peak, it is simple to determine the average grain size of ZnO NPs as a function of peak width (described as the full-width at half maximum peak intensity (FWHM)), peak location, and wavelength.
where λ is the wavelength (Cu Kα), β is the full width at the half-maximum (FWHM) of the ZnO (101) line and θ is the diffraction angle 20 .

Transmission electron microscopy (TEM) analysis
Transmission electron microscopy (TEM) analysis was conducted using JEM-10OCXII TEM (Japan) at 120 kV.The freshly-prepared sample solutions were dropped on carbon coated copper grid to obtain a highly thin film.The ZnO NPs were ready to be investigated after 15 min.

Water vapor and oxygen permeability (WVP&OP)
The water vapor transmission rate (WVTR) was estimated using a GBI W303 (B) Water Vapor Permeability Analyzer (China) using the cup method.According to a standard (ASTM E96) as the total amount of water vapor transported over a unit area in a unit of time under controlled temperature (38 °C) and humidity (4%) conditions.In addition, the gas transmission rate (OTR) was evaluated using a N530 Gas Permeability Analyzer (China) in accordance with ASTM D1434-82 (2003).
L is the slope and the film area is indicated by A (m 2 ).WVP (g mm/m 2 kPa −1 day −1 ) was calculated using Eq. ( 3) L is the film`s thickness (mm) and Pressure is the partial pressures of water vapor in saturated air with 100% relative humidity and 38 °C and air ( Pressure = 5.942 kPa).
where A is the film area (m 2 ), P is the gas partial pressure difference (0.02308 atm, at 25 °C), and f is thickness of the film (m) 21,22 .

Solubility percentage
Solubility was calculated using Eq. ( 6).Film specimens were sliced (2 cm × 2 cm) and weighted (accuracy 0.0001 g), then dried in an oven at 7 °C for 24 h to determine the initial dry mass (M1).The samples were placed in a Petri dish containing 30 mL of water for 24 h at room temperature (25 ± 2 °C) allowing water swelling.Finally, the remaining film specimens were dried in the oven under the same circumstances as previously to determine the final dry mass (M2).For each film sample, two measurements were conducted, and results were expressed as a percentage of the average of the two determinations. 23

Water contact angle measurements (WCA)
The water contact angle of the generated CH/PVA and Zn@TA@CH/PVA films has been determined utilizing a Theata Optical Tensiometer (Data Physics, Model OAC 13EC) (Germany).To calculate the contact angle values, data physics software used the Young fitting procedure on water drop photos collected at the contact point.

Mechanical properties
The thickness of produced films was measured using a thickness dial gauge at three distinct spots, and the mean values were determined.Using electronic universal material testing equipment (Instron 34SC-5 Universal Machine, UK) with a load cell of 5KN, the mechanical characteristics of the films' tensile stress and strain were evaluated.Before testing at a cross head speed of 10 mm per minute, the samples were divided into 80 mm × 20 mm pieces and kept at 23 ± 2 °C and 50 ± 5% relative humidity (RH) for 48 h.The tensile speed was 20 mm/s.Each sample was assessed at least three times, and the average result was noted.Using formula (7), the tensile strength at break (TS) was determined.

DPPH radical scavenging ability
The antioxidant capacity of CH/PVA and ZnO@TA@CH/PVA films of different ZnO@TA loadings was assessed using the 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay (DPPH).A total of 24 mg of DPPH were dissolved in 100 mL of methanol for making the stock solution.Filtration of DPPH stock solution using methanol yielded a usable mixture with an absorbance of around 0.973 at 517 nm 25 .In brief, 4 ml of a 100 mM DPPH methanol solution was added to 20 mg of the film.For one hour, the reaction was kept in the dark.At 517 nm, the absorbency of the reaction solution was measured 26 .
For the ABTS assessment, an ABTS solution was prepared by adding 7 mM of ABTS to 2.4 mM of potassium persulfate solution in a 1:0.5 (v/v) ratio and preserved in the dark for 12-16 h.7 mM of ABTS to 2.4 mM of potassium persulfate solution in a 1:0.5 (v/v) ratio and preserved in the dark for 12-16 h.The ABTS solution was diluted to attain an absorbance of 0.7 (± 0.1) at 734 nm.Similarly, to the DPPH test, about 20 mg of the film sample was immersed in 4 mL of the ABTS solution in the dark for 60 min.The film sample was subsequently picked out to measure the absorbance of the ABTS solution.A pure ABTS solution was used as the control, and the radical scanning activity was analyzed based on the difference in the absorbance of the solution at 734 nm (n = 5).The antioxidant activities of the films were calculated as follows: Accordingly, DPPH radical scavenging activity was obtained (Eq.9): where, A o is the absorbance of the pure DPPH or ABTS solution and A s is the absorbance of the same solution after the immersion of the film 19,27 .

Ultraviolet blocking analysis
The UV-blocking features of CH/PVA and ZnO@TA@CH/PVA films were evaluated by assessing their absorbance and total transmittance from 200 to 2500 nm (UV-Vis-NIR spectrum, Japan).The T (UVA) and T (UVB) of CH/PVA and ZnO@TA@CH/PVA films were estimated using the equations listed below (Eqs.10 and 11), respectively 28,29 .
Where, T λ is the transmittance of TCC/DAC-TH films of the light at the wavelength.

Thermal analysis
Thermo gravimetric assessment have been utilized to evaluate the thermal stability of CH/PVA and ZnO@TA@ PVA/CH films (Universal V4.5A TA Instruments SDT Q600 V20.9 Build 20) under nitrogen.Based on information about weight loss, kinetic investigations were done using the TG curve.The thermodynamic parameters of deterioration were identified.A chemical reaction's rate is commonly assumed (Eq.12).
where, c is the weight of the residual constituents at time t for each fragmentation step, and n is the order of the reaction.Equation ( 13) was utilized to provide further explanation: where, w t is the sample mass at time t and w ∞ is the mass at the end of the fragmentation process.Applying Arrhenius equation to obtain the Eq.(14).
A is the Boltzmann constant, R is the general gas constant (8.314Joules/deg.mole), T is the absolute temperature (Kelvin), and the E a is activation energy.The reaction rate (−dw/dt) could be modified into Eq. ( 15):   www.nature.com/scientificreports/where, w 1 and w 2 are residual amount at times t 1 and t 2 , respectively.The least square approach and n values from 0.0 to 3.0 with 0.5 increments were were used to determine the highest R2, lowest standard error for each n and total activation energies could be estimated 30,31 .

Antimicrobial properties
The agar disc diffusion procedure or the Kirby-Bauer disc diffusion process was exploited to assess the inhibitory investigation of PVA/CH and ZnO@TA@PVA/CH films with different ZnO@TA contents against Staphylococcus aureus, Aspergillus flavus, and Candida albicans.

Statistical analysis
Each experiment was repeated at least three times.Using the "SPSS" application, the results were presented as mean standard deviation (SD).The results were subjected to statistical analysis using the Duncan test to identify variations among treatments at a significance level of 0.05.

Results and discussion
The multifunctional ZnO@TA@PVA/CH films with antibacterial, antioxidant, and UV blocking properties are as-prepared as illustrated in Fig. 1.The possible mechanism of surface modification on ZnO NPs with TA acid involves two proposed mechanisms through formation of hydrogen bonding between -OH of tannic acid with ZnO NPs surface and formation of a π-complex between the surface of ZnO NPs and tannic acid containing phenyl groups through sharing of π-electrons of phenolic rings present in the tannic acid with 4S orbital on Zn +2 via a coordination bonding 32 .
For FTIR spectra of tannic acid show a significant absorption of about 3480-3000 cm −1 with a wide and strong band centered at 3225 cm −1 .This band is corresponded to the broad and strong H-bonded hydroxyl groups (O-H) as well as the C-H bond (aromatic medium).Tannic acid consists of aromatic esters due to the signal properties of carbonyl groups C=O stretching (1699 cm −1 ) and C-O (1313-1187 cm −1 ).The in-plane bending of C-O-H group occurs at 1607-1535 cm −1 , and the out-of-plane bending was assigned to 872 cm −137 .Bands at 1607-1445 cm −1 associated with C-C of aromatic compounds.Various peaks in the 1085-755 cm −1 associated with substituted benzene ring 38 .
For FTIR spectrum of ZnO@TA, the characteristic bands of the ZnO@TA nanoparticles and pristine TA were remarkably comparable, resembling the conventional peaks of TA.It could be claimed that the interaction is pronounced as a π-complex between tannic acid containing phenyl groups and the surface of ZnO NPs.Moreover, the characteristic peaks of aromatic C=C of tannic acid at 1445 cm −1 and 1607 cm −1 were disappeared, indicating the participation of the C=C group in the creation of the coordination complex.This could be due to the interaction of π-electrons of phenolic rings in tannic acid with the 4S orbital on Zn +2 via a coordination link, generating a sandwich-like structure 32 .A significant shift in the band assigned to the hydroxyl groups (O-H) H-bonded and C-H (aromatic medium) at 3225 cm −1 to 3377 cm −1 with somewhat sharpness due to the proposed coordination and hydrogen bonding between ZnO NPs and TA.The out-of-plane bending was assigned to 867 cm −1 shift to 820 cm −1 .These findings indicate that the hydroxyl group of TA could form hydrogen bonding with ZnO 39 .The absorption peak at 414 cm −1 which corresponds to metal-oxygen (ZnO stretching vibrations) vibration mode is strongly observed in ZnO@TA.
For the virgin CH/PVA IR curve has a substantial peak at 3291 due to the overlapping of the -OH and NH 2 groups of PVA and CH, besides a peak at 2861 related to -CH group 28 .These peaks are signatures of polysaccharides and can be witnessed in other polysaccharide spectra 40 .Other peaks at 1649 are due to the C=N amine bond of CH, and at 1561 to NH 3 + , which is formed by NH 2 deformation in acidic conditions.CH 2 bending and CH 3 symmetrical deformations have been verified with bands at 1423 and 1375 cm −1 , respectively.The absorption peak at 1143 cm −1 indicates asymmetric stretching of the C-O-C bridge.The peaks at 1032 and 923 cm −1 indicate C-O stretching. 41which is characteristic of saccharide structure of chitosan.
FTIR spectrum of ZnO@TA@CH/PVA film showed the same spectrum of CH/PVA film with minor shift.The addition of ZnO@TA into CH/PVA causes a minor shift from 3280 to 3292 cm −1 due to hydrogen bonding between CH/PVA and ZnO@TA.The absorption peaks which correspond to metal-oxygen (ZnO stretching vibrations at 414 cm −1 ) of ZnO@TA is powerfully detected in FTIR spectrum of ZnO@TA@CH/PVA indicating the inclusion of ZnO@TA.
The average grain crystallite size Scherer equation of ZnO NPs were estimated utilizing Scherer equation and have an estimated value equaled 1.727 nm.No noticeable peaks of crystalline phase were observed in the XRD patterns of TA, but characteristic two signals (2θ = 11.2° and 24.9°), representing the amorphous nature of TA.
The XRD pattern of the ZnO@TA showed crystalline diffraction peaks with minor shift at 2θ = 32.0°,34.6°, 38.2°, 44.0°, and 56.8° were observed clearly which are indexed by diffraction planes (100), (002), ( 101  patterns of the generated composite films was shifted to 22.4° may be attributable to blending with chitosan.The specific signals of ZnO@TA are not observed in diffractogram of ZnO@TA@CH/PVA indicating that trace amount of ZnO@TA was altered when added to CH/PVA matrix.In order to determine the crystallinity alteration of CH/PVA film when ZnO@TA was added, the crystallinity index of ZnO@TA@CH/PVA was calculated by the following equation (Eq.16): where, A c and A a are crystalline area and amorphous, respectively 44 .
The crystallinity index % of CH/PVA was 67.07%while ZnO@TA@PVA/CH showed a slight increase with inclusion of ZnO@TA given 71.44%.

Transmission electron microscopy analysis
TEM reveals the microscopic structural characteristics of ZnO NPs. Figure 4 shows TEM images of ZnO NPs, where ZnO NPs appear to have a uniform and spherical shape with particle size within the range of ( 16)  www.nature.com/scientificreports/4.95-25.72nm.These results were consistent with that reported by Razieh Galal et al. (2010) 45 .Figure 4 depicts the selected electron diffraction area of ZnO NPs revealing the nano-crystalline structure of ZnO NPs which accords with XRD analysis.

Particle size and zeta potential measurements
Figure 5 shows the Zeta Potential and particle size distribution of ZnO@TA.ZnO@TA NPs have a mean hydrodynamic diameter of 663.8 ± 0.57 nm.Tannic acid aggregation on ZnO NPs arose due to the impact of acidic medium caused by TA molecule 46 , results in the formation of ZnO@TA complex could be the cause of the increased mean hydrodynamic diameter (MHD) of ZnO@TA.Abebe Belay et al. (2015) recently noticed a similar effect.When caffeic acid was added to the ZnO NPs, it was observed that the MHD of the ZnO@caffeic acid NPs increased.When the concentration of ZnO NPs was 7.55 × 10 -6 M, the MHD of ZnO/caffeic acid was 295 nm.When ZnO NPs concentration was 7.55 × 10 -6 and 1.30 × 10 -5 M, respectively, the MHD of ZnO@caffeic acid increased to 396 and 955 nm 47 .The Zeta Potential, which represents the NPs' surface charge, is one significant factor which influences particle stability.If the electric charge on the particle surface is larger, the NPs are not likely to assemble.The Zeta Potential numerical value of ZnO@TA is − 27.83 mV.This indicates ZnO@TA NPs complex are well stabilized due to repulsive forces that prevent aggregations upon aging.The recognized range of the Zeta Potential to provide adequate stability in solution is − 30 to − 20 mV or + 20 to + 30 mV, according to Mojtaba Taghizadeh et al. 39,48 .Almost the same results were reported by Xiaojia He et al. ( 2016), the Zeta potential larger than-25 of TA@TiO 2 NPs 49 .www.nature.com/scientificreports/

Surface morphology
Figure 6 displays SEM image of ZnO@TA as well as mapping and EDAS data.ZnO@TA showed spherical shape in clusters.The existence of ZnO NPs with homogenous distribution in ZnO@TA is further supported by mapping images.In EDAX spectrum, the proportional elemental composition of ZnO nanoparticles in ZnO@TA was confirmed using the energy dispersive X-rays Analysis (EDAX) tool, as illustrated in Figure 6, by measuring the intensity of the characteristic emitted X-rays.In synthesized ZnO@TA nanoparticles, EDAX revealed only the presence of three elements: carbon, zinc and oxygen.The atomic percent compositions of elements are 59.25%, 38.52%, and 2.23% for C, O, and Zn respectively 50 .
Figure 7 shows SEM, EDAX, mapping images of CH/PVA and ZnO@TA@CH/PVA films.PVA/CH film shows uniform smooth surface, without any fracture and air bubbles.The surface image of ZnO@TA@CH/PVA film exhibits surface roughness due to inclusion of ZnO@TA NPs.The proportional elemental composition of CH/PVA film shows three elements namely, C, O, and N with atomic percent 49.89%, 44.41%, and 5.7%, respectively.However, the proportional elemental composition of ZnO@TA@CH/PVA film was 55.27%, 36.97%,5.7%, 2.06% for C, O, N, and Zn, respectively.The mapping images of the film reveal that ZnO@TA nanoparticles are uniformly distributed in ZnO@TA@CH/PVA film matrix as demonstrated in Fig. 7. www.nature.com/scientificreports/

Barrier properties
Table 2 demonstrates the impact of changing ZnO@TA concentrations on barrier properties.WVP measures the rate of moisture that crosses the film, being an important property to be accounted for in packaging applications 51 .
As expected, WVP of ZnO@TA@CH/PVA films tend to diminish with the incorporation of ZnO@TA NPs compared with CH/PVA control film which is generally explained by the physical crosslinking of ZnO@TA NPs nanocomposite, which will diminish the diffusion of water vapor and gases 33 .
The WVP of ZnO@TA@CH/PVA films shows a significant decrease with increasing ZnO@TA concentrations (p˂0.05) in comparison with CH/PVA control.There is no significance difference between PVA/CH films containing ZnO@TA concentrations of 10 and 30 mg however, WVP of ZnO@TA@CH/PVA film containing 50 mg significantly differ than the other films.May be at that concentration (50 mg) is sufficient to cause more crosslinking to CH/PVA films thus restrict mobility of the polymer chains and consequently decreasing the transport of gases.The lowest WVP is 31.98 ± 1.68 g.mm/m 2 .kPa.day which related to ZnO@TA(50)@CH/PVA compared with PVA/CH control film 43.52 ± 1.01 g.mm/m 2 .kPa.day as typed in Table 1.
Other researchers have previously documented the same trend, a study reported by Song et al. ( 2023) indicated that ZnO/plant polyphenols/cellulose/polyvinyl alcohol films showed better water vapor barrier properties Table 2. Influence of ZnO@TA concentrations (mg) WVP and OP of ZnO@TA@CH/PVA film.Superscript letters a-d: different superscripts within the same column indicate significant differences among samples (p < 0.05).than PVA, with a WVP of 12.7 g m −2 h −1 and 10.8 to 7.5 g m −2 h −153 .In another study worth mentioning in which chitosan and tannic acid were crosslinked in neutral and mildly basic circumstances to create chitosan-tannic acid composite films.The pristine chitosan film had strong transmission rates, as evidenced by its WVTR and OTR of 956 g/m 2 ⋅day and 0.39 cc/m 2 ⋅day, respectively.The chitosan-tannic acid composite films, on the other hand, exhibited noticeably lower transmission rates than the native chitosan film because of the formation of a denser structure as a result of physical crosslinks between the two substances 19 .

Solubility of ZnO@TA@CH/PVA film
The influence of ZnO@TA changing concentrations on ZnO@TA@CH/PVA film solubility is shown in Fig. 8.The changing concentration of ZnO@TA has significant impact on solubility of the films (P < 0.05).The solubility of ZnO@TA@CH/PVA films drastically tend to decrease with increasing loadings of ZnO@TA.The solubility percentages are 64.35 ± 2.4 %, 47.65 ± 2.2 %, 36.68 ± 1.1%, and 30.36 ± 1.4% that are belonged to CH/PVA, ZnO@ TA(10)@CH/PVA, ZnO@TA(30)@CH/PVA, and ZnO@TA(50)@CH/PVA films.These results may be ascribed to crosslinking reasons and development of more hydrogen bonding between ZnO@TA and functional groups of base film components as mentioned before.

Water contact angles measurements (WCA)
Figure 9, shows the WCA of CH/PVA and ZnO@TA@CH/PVA films containing different concentrations of ZnO@TA.According to a general theory, a contact angle that is smaller (below 90°) indicates a material's hydrophilic character, whereas a greater value (above 90°) shows a material's hydrophobic nature.The contact angle of CH/PVA film is 76.6°.For ZnO@TA concentrations of 10, 30, and 50 mg, respectively, the contact angles of  www.nature.com/scientificreports/ZnO@TA@CH/PVA films are increased by 7.18%, 19.19%, and 33.42%, respectively.These findings may due to ZnO@TA addition-induced roughness to the surface of ZnO@TA@CH/PVA films occurred.

Mechanical properties
Table 3 depicts tensile strength and elongation % at break of ZnO/TA@ PVA/CH as a function of increasing ZnO@TA concentrations.The tensile strength of neat PVA/CH film was 30.47 ± 0.96 MPa.As the loading of ZnO@TA increase, tensile strength of ZnO@TA @PVA/CH composite films significantly increases (p < 0.05).
Tensile strength of ZnO@TA @PVA/CH composite films are 37.47 ± 1.09, 43.33 ± 1.21, and 48.72 ± 0.23 MPa up to 10, 30, and 50 mg of ZnO@TA loadings, respectively.That enhancement in tensile strength may be attributable to stronger intermolecular forces that can occur between the polymer chains (PVA/CH) and ZnO@TA.When hydroxyl groups of PVA/CH and ZnO@TA nanoparticles interact, covalent and hydrogen bonds developed due to presence of extensive tannic acid hydroxyl moiety, thereby increasing the molecular force.The development of cross links between polymer chains was facilitated by the ZnO@TA.Therefore, films containing ZnO@ TA nanoparticles had a more compact film matrix structure, which resulted in the development of stronger films 16,19 .This tensile enhancement is agreed with results reported by Aswathy Jayakumar et al. ( 2023), active and intelligent composite films based on polyvinyl alcohol, chitosan, zinc oxide nanoparticles, and sweet purple potato extract.The tensile strength of the chitosan/polyvinyl film was 13.0 MPa and increased to 30.8 MPa for chitosan/polyvinyl film composite film containing zinc oxide nanoparticles, and sweet purple potato extract 54 .Youngs modulus of ZnO@TA @PVA/CH composite films significantly increases (p < 0.05).Young`s modulus of ZnO@TA @PVA/CH composite films are 1436.66 ± 47.5, 1905.52 ± 29.3, and 2163.46 ± 61.4 MPa up to 10, 30, and 50 mg of ZnO@TA loadings, respectively compared with control 1034.32 ± 52.8 MPa.
Additionally, table 3 shows the effects of ZnO@TA changing loadings on the elongation at break of PVA/ CH composite films.As can be seen, the percentage of elongation at break significantly decreased with increasing of ZnO@TA concentration (p < 0.05).Despite the increased increments, there is no significant difference between control film and PVA/CH containing 10 mg ZnO@TA.PVA/CH film showed an elongation at break of 35.10 ± 2.3%, whereas the nanocomposites with 10, 30 and 50 mg ZnO@TA showed an elongation at break of 32.54 ± 1.07, 27.02 ± 2.19, and 19.62 ± 2.3%, respectively.This is because the rigidity of matrix increased by adding ZnO@TA.Moreover, strong interaction between ZnO@TA and PVA/CH chains could restrict chain movements and consequently blocks its ability to flow and reduce its ductility 55 .Further, the significant improvement in tensile strength accompanied with decreasing in elongation at break which can be translated as ZnO@TA NPs addition can behave as an efficient reinforcing agent.Notably, the distribution of nanofillers and their interaction with the polymer matrix are strongly correlated with the reinforcing effect in nanocomposite films 56 .Considering the EDAX image in Fig. 7, the significant tensile improvement can be attributed to the homogeneity dispersion of ZnO@TA NPs within CH/PVA film forming strong interfacial adhesion which minimizes phase separation and allows efficient stress transfer at the interface 57 .Indeed, the addition of ZnO@TA NPs in the CH/PVA film matrix enhances mechanical performance.In 2023, Su Jin Lee and colleagues created a multifunctional chitosan (CH) film that contains 0.5-1.0weight %of tannic acid (TA).Tris buffer (pH 8.5) and phosphate-buffered saline (CH-TA/P) were used to neutralize the chitosan films.The virgin CH's tensile strength and elongation % at break were 58.1 ± 5.1 MPa and 8.1 ± 1.8%, respectively.On the other hand, CH-TA0.5/P,CH-TA0.5/T,CH-TA1.0/P, and CH-TA1.0/Tfilms had significantly greater tensile strengths at 109.8 ± 4.9, 134.0 ± 5.1, 63.0 ± 8.1, and 112.8 ± 8.2 MPa, respectively 19 .

Antioxidant activity
DPPH free radicals scavenging activity, in which DPPH functions as a reducing agent or electron donor, is one of the frequently used techniques for determining antioxidant activity.In the presence of antioxidants, DPPH radicals change from a dark violet color to a transparent color and the absorbance at 517 nm is measured to determine the proportion of DPPH antioxidant activity.Figure 10 depicts the percentage of free radical-scavenging Figure 10.DPPH scavenging ability % of ZnO@TA@PVA/CH films as a function of ZnO@TA concentrations.(a-h) Non-identical letters denote statistical difference (p < 0.05).activity of ZnO@TA@PVA/CH films as a function of ZnO@TA concentrations.ZnO@TA@PVA/CH films show a significant increasing in DPPH antioxidant activity in the developed ZnO@TA@PVA/CH films with increasing ZnO@TA when compared control film (p < 0.05).The DPPH radical-scavenging activity % of ZnO@TA@PVA/ CH films increased gradually to 50.0 ± 2.7%, 60.21 ± 4.0%, and 69.35 ± 1.6%, respectively with the increasing concentration of ZnO@TA from 10 to 50 mg, respectively compared with PVA/CH film (11.29 ± 3.2%).The PVA/ CH control film reveals a moderate scavenging activity.However, the increasing in antioxidant activity could be due the galloyl groups in tannic acid may contribute to its potent hydrogen and electron-donating properties 58 .Similarly, Fig. 10 the ABTS radical scavenging ability.The antioxidant activities of CH/PVA, ZnO@TA(10)@CH/ PVA, ZnO@TA(30)@CH/PVA, ZnO@TA(50)@CH/PVA films were 32.29 ± 1.1%, 70.0 ± 1.23%, 83.21 ± 1.61%, and 90.95 ± 2.12%, respectively.ZnO@TA@CH/PVA film has superior antioxidant activity towards ABTS more than DPPH radicals.Previous study reports have documented the same phenomenon, a novel antioxidant-containing zinc oxide (ZnO) nanoparticle was created by immobilizing the antioxidant 3-(3,4-dihydroxyphenyl)-2-propenoic acid, also known as caffeic acid, CA), on the surfaces of ZnO nanoparticles treated with micro-dielectric barrier discharge (DBD) plasma.ZnO@CA nanoparticles efficiently scavenged ABTS radicals at concentrations ranging from 20 to 100 µM, with activity ranging from 44.99 to 73.68%, respectively 17 .Also, research has been done on the polyvinyl alcohol-based film's antioxidant capacity when lignin nanoparticles loaded with potassium sorbate (LNP@PS).Pure PVA is not capable of scavenging DPPH.However, the color of the mixture of DPPH and film extracts steadily lightened and eventually turned orange when the ratio of LNP@PS and/or TA added to the composite film increased.This suggested that there was strong antioxidant activity in the composite films.The intensity of the DPPH absorption peak was significantly reduced and the free radical scavenging activity (RSA)   www.nature.com/scientificreports/increased to 50.9% after only 1% of LNP@PS (LNP@PS-1-TA-0) was added to PVA.With LNP@PS-3-TA-5, the ideal RSA value of 92.6% was attained 59 .

UV-shielding properties
Food packaging transparency is an essential concern in packaging materials, and it has a clear impact on consumer choices 5,60 .Food shelf life can be reduced by microbial development and/or biochemical processes like oxidation.Exposure to UV light can accelerate lipid oxidation in packaged food, which can cause food to deteriorate.Thus, foods, especially those with high fat content, require antimicrobial packaging materials with potent UV light blocking capabilities property 61 .Although PVA is the most often used packaging material, it has poor UV-shielding qualities.However, PVA UV-shielding performance has been improved by mixing it with biomaterials and nanoparticles 62 that improve UV absorbance.Furthermore, adding a third phase to the PVA/ biomaterials mixture, such as nanomaterials filler, could improve the UV-blocking properties 9,63 .The optical characteristics of the PVA/CH and ZnO@TA @PVA/CH films have been assessed using digital images Fig. 11A.
As the ZnO@TA content increased, all of the ZnO@TA @PVA/CH films turned pale brown.Figure 11B depicts the UV transmittance of neat PVA/CH and ZnO@TA @PVA/CH films using various ZnO@TA NP ratios which illustrate the effect of ZnO@TA NP ratios on the UV-shielding properties of the PVA/ CH films.Corresponding to the equations the transmittance of UV-A (320-400) and UV-B (280-320) were implemented to explore UV Shielding properties.The virgin PVA/CH film exhibits an excellent UV shielding capacity where UV transmittance of UVA (91.414%) and UVB (99.198%) and that can be attributed to hydrogen bonding as a HOMO-LUMO interaction between PVA and CH.
Multiple investigators have already documented similar behaviors, for example, the biocomposite ZnO/plant polyphenols/cellulose/polyvinyl alcohol film was created.According to the light shielding study, the cellulose/ PVA film contained 1wt% of ZnO/polyphenol mixture can virtually completely filter UV and visible light 53 .In a nother investigation, lignin nanoparticles loaded with potassium sorbate (LNP@PS) as additives to polyvinyl alcohol-based active packaging films have been explored.Pure PVA films show terrible UV-shielding performance but outstanding optical transparency, with transmittance ratios of 80%, particularly in the UVA and UVB regions, and 90% in the visible light range.The UV shielding performance increased from 96.78 to 989.99% when comparing film containing 3% LNP@PS/3% TA and 3% LNP@PS/5%TA, and from 94.79 to 989.99% when comparing with film containing 1% LNP@PS/5%TA and 3% LNP@PS/5%TA, respectively.Both showed a notable decrease in UV transmittance 59 .

Thermal decomposition analysis studies
The thermodynamic properties of CH/PVA and ZnO@TA@CH/PVA composite films were investigated using thermogravimetric analysis, as illustrated in Fig. 12.The initial weight loss for all the produced films took place between 25 and 130 °C, which might be attributable to the loss of absorbed water molecules, notably the weight loss increased after adding ZnO@TA attributed to the degradation of tannins small molecules such as CO, CO 2 , and phenol from high molecular weight macromolecules into smaller chain fragments 64 .The initial decomposition temperature of ZnO@TA @CH/PVA composite film was slightly elevated to 242 °C when contrasted with CH/PVA film (235 °C) with weight loss percentage.The rise in early degradation temperature could be related to the inclusion of ZnO@TA nanoparticles as shown in Table 5.
CH/PVA film exhibits three-stage decomposition however, ZnO@TA @CH/PVA film has four degradation steps.The major second thermal decomposition step for CH/PVA is in the temperatures range 136.45-278.14°C was attributed to the thermal decomposition of chitosan by deacetylation 4 .ZnO@TA @CH/PVA film sifts to higher temperature range (142.54-304.0°C) and that may be attributed to the thermal stability of ZnO@TA@CH/ PVA film films being better than CH/PVA film after addition of ZnO@TA NPs (Table 4).This temperature range is linked to the dehydration of the saccharide rings, depolymerisation, and decomposition of the polymer units 65 .
And the maximum weight loss rate temperature was slightly increased with the addition of ZnO@TA content from 42.77 wt % to 67.0 wt.% owing to the strong interfacial interactions between the functional groups of ZnO@ TA and the macromolecular chains of CH/PVA.
The thermal decomposition of PVA as per previous report 66,67 occurred between 391.72 and 489.43 °C and 404.03-459.97°C for CH/PVA and ZnO@TA @CH/PVA respectively.The third decomposition range of ZnO@ TA @CH/PVA is between 303.65 and 373.79 °C which could be attributed to two reasons the first is the depolymerisation/hydrolysis of tannic acid 64 and the second is the thermal degradation of cross-linked bond (hydrogen bond) forming via tannins polyols occurred with PVA and chitosan beside to the degradation of zinc oxide nanoparticles of ZnO@TA @CH/PVA film (Table 4).Previous research has shown that zinc oxide nanoparticles can operate as thermal insulators by limiting the mobility of polymer chains.This demonstrated the thermal stability of the generated composite films and was consistent with prior results 68 .The knowledge of E a allows detecting reaction mechanism over a wide temperature range.The total activation energy (Ʃ E a ) of ZnO@TA @CH/PVA is higher than CH/PVA.The Ʃ E a are −0.02803K J mol− 1 and −0.03620K J mol −1 for CH/PVA and ZnO@TA@CH/PVA, respectively (Table 4).This result indicates that ZnO@TA @CH/PVA film is more thermally stable than CH/PVA film.

Antimicrobial studies
Figure 13 shows the antimicrobial activity against Gram-Positive Bacteria: Staphylococcus aureus, pathogenic yeast Candida albicans and crops pathogen: Aspergillus flavus.All films show antimicrobial influence against all tested microorganisms.As shown in Table 5, the inhibition zones (mm) significantly increase with increasing loadings of ZnO@TA, the effective concentration of ZnO@TA is 50 mg at which ZnO@TA@CH/PVA film exhibits the largest inhibition zone (11 ± 1.0 mm) against Staphylococcus aureus compared with amoxicillin/clavulanic positive control (26 ± 1.0 mm) and 12.3 ± 0.57 mm and 13.6 ± 0.57 mm for Aspergillus flavus and Candida albicans, respectively compared with clotrimazole fungal positive control 15.0 ± 1.0 mm and 14.6 ± 0.57 mm, respectively.Several studies have demonstrated the efficacy of zinc oxide nanoparticles for bacterial membrane disruption, enzymatic inhibition, interaction with genes and proteins, and protein inactivation 69 .Previous studies have shown comparable behavior, Kyong-Hoon Choi et al. ( 2017) have reported preparation of a new zinc oxide (ZnO) nanoparticle with antioxidant capabilities.The ZnO nanoparticles were treated with micro-dielectric barrier discharge (DBD) plasma to immobilize the antioxidant 3-(3,4-dihydroxyphenyl)-2-propenoic acid (caffeic acid, CA).ZnO@CA nanoparticles shown strong antibacterial action against Escherichia coli and Staphylococcus aureus, including resistant strains like methicillin-resistant S. aureus 17 .Also, cellulose/ PVA film containing (1.0 wt% zinc oxide/plant polyphenols was created by Da Song and Li-Wei Ma et al. ( 2023) through straightforward hydrothermal and casting techniques.The study evaluated the antibacterial activities of against Escherichia coli and staphylococcus aureus, achieving 4.4 and 6.3 mm inhibition zones, respectively 53 .A study has been reported by Denice S. Vicentini et al. (2010) has prepared ZnO nanoparticles have been produced from polyester using the Pechini method, which involves reacting citric acid and ethylene glycol.The resulting ZnO nanoparticles were then mixed with varying concentrations of polyoxyethylene sorbitan monooleate, or Tween 80 (T80), to create blend films of chitosan (CH) and poly (vinyl alcohol) (PVA).When the films' antibacterial activity was evaluated, the ZnO nanoparticle-containing films demonstrated antibacterial activity against the bacterium species Staphylococcus aureus.Without ZnO nanoparticles added, the S. aureus species microorganisms in the films continued to be viable; but, when exposed to ZnO nanoparticles, they ceased to be viable.Consequently, these findings imply that the ZnO nanoparticles' presence is what causes the antibacterial activity 11,70 .Another study examined the efficiency of chitosan-coated film with varied quantities of Moringa oleifera seed powder (MOSP) as reinforcement agent and tannic acid (TA) as a crosslinker reported by Raja Venkatesan et al. (2024).The biocomposite films, with 10.0 wt.% MOSP content, showed increased antimicrobial and antifungal activity against bacteria like Staphylococcus aureus, E. coli, A. niger, and Candida albicans, making them ideal food packaging materials 71 .

Conclusion
Active packaging film ZnO@TA@PVA/CH with different ratios of ZnO@TA nanocomposite was successfully created.The structure of the prepared films was characterized using FTIR, and XRD which revealed the existence of ZnO@TA nanocomposite in the PVA/CH matrix film.Moreover, the morphological structure of ZnO@ TA @PVA/CH composite film was figured using TEM, and SEM which showed crystallites characteristic of nanostructured ZnO@TA, in addition.The mapping images of the film reveal that ZnO@TA nanoparticles are uniformly distributed in ZnO@TA@PVA/CH film matrix.The results also showed that increasing ZnO@TA content enhanced the thermal stability, water vapor barrier properties, antibacterial and mechanical properties of PVA/CH films.Besides, increasing ZnO@TA nanocomposite content showed a significant increase in DPPH antioxidant activity and UV-Shielding properties.ZnO@TA @PVA/CH film has a broad antimicrobial spectrum action against Gram-Positive Bacteria: Staphylococcus aureus, pathogenic yeast Candida albicans and crops pathogen: Aspergillus flavus.The maximum inhibition zone was created by ZnO@TA(50)@PVA/CH film.In light of the previous results, incorporating ZnO@TA nanocomposite in the PVA/CH matrix could be a promising way for efficient biodegradable packaging material.
The demands of the modern world cannot be adequately and successfully met by traditional packaging.To reduce pathogens resistance in food, active packaging will grow in the future due to consumer preferences minimally processed and naturally preserved products as well as the food industry's need for significant investments in the quality and safety of its products.Additional research is required in a number of active-packaging system domains for this purpose.To improve film characteristics, future studies should look at large-scale production and the incorporation of additives such natural extracts and nanoparticles.Films made of a chitosan/ PVA combination provide a viable, environmentally friendly substitute for plastic packaging manufactured from petroleum, and they may find use in actual food preservation.
Rate = dc/dt = kc n

(
), (102), and (110) of ZnO NPs(Li, et al. 2016).The X-ray diffraction profiles of CH/PVA and ZnO@TA@CH/PVA films are shown in Fig.3.The XRD profile of CH/PVA and ZnO@TA@CH/PVA films are very similar.The CH/PVA film diffractogram exhibits peaks in 2 Theta = 16.9°,23.2°, and 34.6°where amorphous diffraction peaks at 2 Theta = 23.2° is indicative of the blending of CH/PVA43 .The 2θ value at 19.43° revealed the crystal structure of PVA, which was found across all diffraction

Figure 1 .
Figure 1.A schematic diagram of suggested mechanism of ZnO@TA formation and construction of ZnO@ TA@CH/PVA films.

Figure 4 .
Figure 4. TEM and selected electron diffraction area of ZnO.

Figure 8 .
Figure 8.Effect of ZnO@TA changing concentrations on ZnO@TA@ CH/PVA film solubility.a-d: different superscript letters represent significant difference at 5% level of probability (P < 0.05).

Table 4 .
Thermal degradation kinetic parameters of CH/PVA and ZnO@TA @CH/PVA films.